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LIBRARY

Michigan Sm
University

 

ABSTRACT

AN ASSESSMENT OF THE TAXONOMIC RELATIONSHIP
BETWEEN SPOROTHRIX SCHENCKII HEKTOEN AND
PERKINS AND CERATOCYSTIS STENOCERAS
(ROBAK) C. MOREAU

 

BY
Ronald Anton Heinrichs
Recently, researchers have noted marked similarities

between conidial states of members of the genus Cerato-

cystis, pathogenic on plants, and §porothrix schenckii

 

Hektoen and Perkins pathogenic on humans and animals. It

was hypothesized that g. schenckii is an asexual relative

 

of the genus Ceratocystis. This relationship was tested

 

using four strains of g. schenckii, isolates of a perfect

 

and imperfect stage of Ceratocystis stenoceras (Robak) C.

 

Moreau, its pathogenic mutant, strain of g. fagacearum, and

a strain of Calcarisporium arbuscula.

 

Acrylamide gel electrOphoresis was used to analyze
the soluble proteins extracted from the fungi. The gels
were stained to show the protein bands and duplicate gels
were treated to locate acid and alkaline phosphatase
activities. The liquid culture medium and mycelial

extracts were also assayed for acid and alkaline

Ronald Anton Heinrichs

phosphatase activity. Sugars hydrolyzed from the cell
wall polysaccharides of the fungi were analyzed by paper
chromatography. The effect of thiamine on the growth of
Q. stenoceras was also tested.

Results from electrophoresis indicated, from the
number of matching protein bands and sites of acid

phOSphatase activity, that g. schenckii was similar to

 

Q. stenoceras. The level of acid phosphatase activity

 

was much higher than the alkaline phosphatase activity in
the culture liquid at 24 C as well as in the protein
extracts for most of the strains. The protein extracts
showed similar results for enzyme activity. Both fungi
showed the presence of mannose and rhamnose in the cell

wall polysaccharides. Ceratocystis stenoceras and

 

g. schenckii appeared to require thiamine for growth.

The hypothesis that g. schenckii is a relative of

 

the genus Ceratocystis is supported by the results of

this study.

AN ASSESSMENT OF THE TAXONOMIC RELATIONSHIP
BETWEEN SPOROTHRIX SCHENCKII HEKTOEN AND
PERKINS AND CERATOCYSTIS STENOCERAS
(ROBAK) C. MOREAU

 

 

BY

Ronald Anton Heinrichs

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Botany and Plant Pathology

1971

TO MY WIFE KATHLEEN

ii

ACKNOWLEDGMENTS

The author is pleased to express great appreciation
and sincere thanks to Dr. E. S. Beneke and Dr. A. L. Rogers
for their encouragement and guidance throughout his
graduate program.

Dr. C. J. Pollard gave of his materials and advice
which was sincerely appreciated and very helpful in the
completion of this study. Dr. W. G. Fields deserves
sincere thanks for his inspiration and helpful suggestions.

The author is very grateful to Drs. F. Mariat and
H. L. Barnett for providing strains of Ceratocystis

stenoceras and Calcarisporium arbuscula.

 

To his wife, Kathleen, the author is deeply
grateful. Her patience, understanding, and assistance

made the completion of this study possible.

iii

TABLE OF CONTENTS

 

 

 

 

 

Page
DEDICATION O O O O O O O O O O O O O 0 ii
ACKNOWLEDGMENTS . . . . . . . . . . . . iii
LIST OF TABLES O O O O O O O O O 0 O O 0 Vi
LIST OF FIGURES . . . . . . . . . . . . Vii
INTRODUCT ION O O O O O O O O O O O O O 1
REVIEW OF LITERATURE . . . . . . . . . . . 2
MATERIALS AND METHODS . . . . . . . . . . 6
Culture Media . . . . . . . . . . . . 6
Extraction of Solubl Protein . . . . . . . 7
Disc Electrophoresis Procedure . . . . . . . 8
Location of Acid and Alkaline Phosphatase Activity
in the Gels . . . . . . . . . . . . 10
Survey for Enzymatic Activity of the Organisms in
Liquid Media and in Protein Extracts . . . . ll
Extracting and Analyzing Mycelial Polysaccharides. 12
Requirement of Thiamine for Growth of g.
stenoceras 1013 and 1020 . . . . . . . . 13
RESULTS 0 O O O O O O O O O O O O O O 14
Disc Electrophoresis of Soluble Protein Extracted
from the Mycelium of S. schenckii, Q. stenoceras,
Q. fagacearum, and Calcarisporium arbuscula . . 14
Tests for the Presence of Acid and Alkaline
Phosphatase Activity in the Gels . . . . . 18
Survey for Acid and Alkaline Phosphatase Enzyme
Activities in Culture Filtrates and Protein
Extracts . . . . . . . . . . . . . 20
Sugars Derived from Fungal Polysaccharides . . . 22
Effect of Thiamine Deficiency on Growth . . . . 22

iv

DISCUSSION

SUMMARY .

BIBLIOGRAPHY

Page

27

35

37

LIST OF TABLES

Table Page

1. Average Rf values of protein bands (six
replicates) in the electrOphoretic patterns
of protein extracts of eight organisms in
standard medium for seven days at 24 C . . . l6

2. The number of matching protein bands between
pairs of gels of four strains of g. schenckii,
two strains of g. stenoceras, and one each of
Q. fagacearum, and g. arbuscula . . . . . 18

 

 

 

 

3. Acid and alkaline phosphatase activities in the
filtrate of eight fungi grown in standard
liquid medium for seven days at 24 C . . . . 21

4. Acid and alkaline phOSphatase activities in the
soluble protein extract of the mycelium of the
eight fungi in standard liquid medium for
seven days at 24 C . . . . . . . . . . 21

5. Requirement of thiamine for growth. Weight
(g.) of mycelium from seven day liquid shake
culture dried at 96 C . . . . . . . . . 26

6. Coefficients of similarity . . . . . . . . 31

vi

LIST OF FIGURES

Figure Page

1. ElectrOphoretic patterns in acrylamide gels
from soluble protein extracts of S.
schenckii strains. (a) Pulmonary, (b) Flint,
(c) Belo Horizonte, (d) Sparrow, S.
stenoceras strains, (e) 1013, (f) 1020,
S. fagacearum, (g) fag, and S. arbuscula,
(h) arb. . . . . . . . . . . . . . l7

 

 

 

2. ElectrOphoretic gels of S. schenckii (Pulmo,
Flint, Belo, Spar), S. stenoceras (1013,
1020), S. fagacearum (fag , S. arbuscula
(arb). Sites of acid phosphatase activity
with Rf value . . . . . . . . . . . 19

 

 

 

 

3. Paper chromatography of sugars derived from
mycelium of S. soggnckii strains Pulmo (P)
and Belo (B), and S. stenoceras 1013 (13) . . 23

 

 

4.. Paper chromatography of sugars derived from
mycelium of S. stenoceras 1020 (20) and
S. fagacearum (fag) . . . . . . . . . 24

 

 

5. Paper chromatography of sugars derived from
mycelium of S. schenckii strains Pulmo (P)
and Belo (B), S. stenoceras 1013 (13) and
1020 (20), and S, fagacearum (fag) and each
sample combined with mannose and rhamnose . . 25

 

 

 

vii

INTRODUCTION

Sporothrix schenckii Hektoen and Perkins 1900
produces a chronic disease, sporotrichosis, involving the
subcutaneous tissue in man and animals. The relationship

of Ceratocystis sp. to Sporothrix schenckii was studied by

 

Mariat et al. (1968) and Mariat (1969, 1970), who pro-
posed the working hypothesis that the conidial form of

Ceratocystis sp. represents the ancestral form of S.

 

schenckii. This hypothesis was tested by Taylor (1970)

 

who compared cultural, morphological, and serological

characteristics and mouse virulence of several Ceratocystis

 

species in the conidial state with several strains of

S. schenckii. His results supported the hypothesis of

 

Mariat.
The objective of this investigation was to compare

the relationship between selected strains of Ceratocystis

 

and Sporothrix and Calcarisporium, a genus closely related

 

 

to SporothriX, using polyacrylamide gel electrOphoresis,
acid and alkaline phosphatase enzyme activity assays,
paper chromatography of sugars hydrolyzed from mannan
containing polysaccharides, and growth requirements for

thiamine.

 

 

(Ill. ‘l’ [’l

REVIEW OF LITERATURE

Sporothrix schenckii Hektoen and Perkins 1900 is a
dimorphic fungus able to grow in culture at 24 C as the
mycelial phase, which converts to the yeast phase at 37 C
on media supplemented with an organic nitrogen source and
a C02 tension of five percent. The organism at both
phases requires the addition of thiamine to the medium for
growth. The yeast phase produces the disease sporotrichosis
which occurs in man and animals and is characterized by
nodular lesions in the lymph system and skin or subcutaneous
tissues of the hands, arms or legs. A disseminating form
of the disease may attack the skeletal structure or internal

organs. The mycelial phase at 24 C has branching, septate

hyphae about 2p in diameter. The hyaline, non-septate
conidia range in shape from spherical to.pyriform and
rarely triangular, appearing on short sterigmata, singly
along the hyphae or more often as several conidia clustered
on the end of a conidiOphore (Beneke and Rogers, 1970).

The species was first isolated from a human case
and described by Schenck (1898) and tentatively placed in

the genus Sporotrichum by E. F. Smith. Later, Hektoen and

 

Perkins (1900) isolated the same organism from a human
case and assigned it to a new genus calling it Sporothrix

schenckii. This new classification was ignored by other

 

2

workers in the field who continued to use the previous

generic name, Sporotrichum, and added different species

 

names .

A revision of the nomenclature of Sporotrichum

 

schenckii began with the effort of Hughes (1958) in regard
to the classification of the Hyphomycetes. Part of his work

dealt with the genus Sporotrichum as described by Link

 

(1809). Eleven of the 12 type species of Sporotrichum

 

were found to be unrelated and could be classified else-
where leaving only S. aureum as the lectotype species.
This finding was supported by the views of Carrion and
Silva (1955) using 1821 as the starting point for nomen-
clature of the Hyphomycetes. Carmichael (1962) then
found Sporotrichum schenckii to be unrelated to the
lectotype species of the genus and placed it in the genus

Sporothrix as originally proposed by Hektoen and Perkins as

 

S. schenckii Hektoen and Perkins 1900. He also noted that

 

S. schenckii bears its closest resemblance to Calcarisporium

 

 

Preuss. Barron (1968) mentioned that S. pallidum bears a

 

closer resemblance to S. schenckii than it does to S.

 

arbuscula, the type species of the genus. The organism

 

also appears similar to the "Sporotrichum" states found

in some species of Ceratocystis.

 

Recent deve10pments have put the present classifica-
tion in question again. Mariat et a1. (1968) isolated

seven strains of Ceratocystis sp. which had morphological

 

 

 

lll'lnflll.‘ [f

and physiological characteristics similar to nonpigmented
strains of S. schenckii. Perithecia were produced in
culture after one month, enabling positive identification

of the genus of these fungi. These strains were not
pathogenic for the mouse or hamster. Mariat (1968) observed
that members of the two genera were found in identical
habitats, isolates of both genera formed identical mycelial

structures, which in the case of Ceratocystis isolates

 

went on to form perithecia, and Sporothrix occasionally

 

formed nonpigmented conidia on synnemata the same as
Ceratocystis in its form-genus Graphium. Mariat (1969)
later discovered one of the isolates Ceratocystis stenoceras
(Robak) C. Moreau, had developed pathogenicity after one
passage through a hamster but it no longer produced peri-
thecia. On this basis, Mariat (1970) developed a working
hypothesis that the Sporothrix-like conidial form of

Ceratocystis is an ancestral form of S. schenckii.

 

Taylor (1970) expanded on the investigations of
Mariat. He compared the conidial states of several

species of Ceratocystis with strains of S. schenckii and

 

 

found the cultural, morphological, serological character-
istics, and virulence for mice to be essentially the same.

Certain Ceratocystis species in the conidial states

 

appeared identical t°.§- schenckii and could only be

 

identified pr0per1y by the formation of the perfect stage.

The technique of polyacrylamide gel disc electro-
phoresis was used by deBievre (1967) to study the yeast and

mycelial phases of S. schenckii. This method has also been

 

used by Schechter §E_2l- (1966), Stipes (1970), and
Sorenson EE_El° (1971) in their efforts to determine the
taxonomic status of fungi. The value of this procedure as
a taxonomic tool, however, has been questioned by Sorenson
§£_3l. (1971) because of variability of protein patterns
between strains of the same species. They have used
mathematiCally similar coefficients to determine the degree
of relationship between fungi based on the results of disc
electrophoresis. In addition to the use of strains (to
detect proteins in the gels), Stipes (1970) detected sites
of alkaline phosphatase activity utilizing the methods

of Davenport (1960) and Jensen (1962). The enzyme results
provided an additional comparative basis for the classifica-
tion of organisms.

On the basis of the enzyme work by Beneke §E_El-
(1969), and Rogers and Beneke (1971), demonstrated that
the level of enzyme activity in culture filtrates differed
in the strains of organisms tested. This may help to
differentiate strains of fungi.

Proton magnetic resonance spectra of the mannose-
containing polysaccharides and their component sugars
identified by paper chromatography were used by Spencer
and Gorin (1971) as a means of grouping various species of

Ceratocystis and the form genus Graphium.

 

MATERIALS AND METHODS

Culture Media

 

Stock cultures were obtained from the following

sources: four strains of S. schenckii (Spar, Flint, Pulmo,

 

and Belo), and one of Ceratocystis fagacearum (fag) from the

 

Michigan State University stock culture collection;
Ceratocystis stenoceras strains 1013 and 1020 from Dr. F.

Mariat, Pasteur Institute, Paris; and Calcarisporium

 

arbuscula Preuss (Arb) from Dr. H. L. Barnett, West
Virginia University. The organisms were cultured in 250 ml.
Erlenmeyer flasks with 100 ml. of liquid medium. The
medium of deBievre (1967) was used with a modification in
the trace element solution. The composition of the medium
is: potassium phosphate, 0.91 g; sodium phosphate-7 H20,
1.80 9; magnesium sulfate, 0.60 g; potassium chloride,

1.00 g; l-arginine monohydrochloride, 1.00 9; glucose,

9

20. g; 1x10-5M thiamine hydrochloride; 1x10- M biotin; and

1 m1. of a trace element solution composed of ferric

chloride-6 H O, 1.0 ug.; 0.5 ug. each of manganese

2

chloride-4 H20 and cobalt sulfate-7 H2

0.8 ug.; made up to a liter of water. For solid medium

O; and zinc chloride,

15 g of agar was added. This medium was then inoculated

with the fungi. The inoculum consisted of 5 ml. of a cell

suspension made by taking three plugs, one cm. in diameter
from the edge of an actively growing colony, on solid medium
at 24 C; adding the plugs to 50 ml. of liquid medium and

run in a Waring blender for 30 seconds. The medium was of
the same composition as noted above. Each fungus was
cultured in two flasks at 24 C on a rotary shaker at 160

rpm.

Extraction of Soluble Protein

 

After seven days the flasks were checked micro-
scopically for contamination, the mycelium collected by
filtration on two sheets of Whatman No. 1 filter paper in
a Buchner funnel under vacuum and washed three times with
distilled water. The mycelial mats were added to 40 m1.
of acetone at -20 C and 3 gm. of acid washed sand in a
Servall Omni-mixer which was run for three minutes at
15,000 rpm with the container immersed in an acetone-dry
ice bath. The resulting slurry was filtered on Whatman
No. 1 filter paper in a Buchner funnel under reduced
pressure, washed with 50 m1. of acetone at -20 C, and
worked to a dry powder with a spatula. The powder was
stored at -20 C. In preparation for electr0phorsis,

50 mg. of powder were added to 1 ml. of 0.004 M sodium
bicarbonate solution and stored overnight at 4 C. The
suspension was centrifuged at 4500 rpm for 25 minutes at
4 C and the supernatant solution collected and analyzed

for the amount of protein following the Lowry method

(Lowry et a1., 1951, as modified by Miller, 1959). The
protein extract was either used immediately or stored at
-20 C in 750 pg portions with 0.1 m1. of 40% sucrose

solution.

Disc Electrophoresis Procedure

 

The method of disc electrOphoresis described by
Davis (1964) was followed, except for the use of a 12%
lower gel and TRIS-glycine buffer diluted three times.
Polyacrylamide gel columns were made in 5 x 100 mm.
cylindrical glass tubes. The gels were composed of two
layers. The lower layer was a small pore gel where
separation of the proteins took place. The upper layer
was a large pore gel where the proteins became stacked
according to size and charge before being separated in the
lower gel. Materials used in making the gels were acryla-
mide, N-N'-methy1enebisacrylamide (BIS), tris aminomethane
(TRIS), N,N,N'-tetramethylethylenediamine (TEMED),
riboflavin, hydrochloric acid, and ammonium persulfate.

Six stock solutions were prepared as follows:

Stock A Stock B
1 N HCl 48 m1. 1 N HCl approx. 48 m1.
TRIS 36.6 g. TRIS 5.98 g.
TEMED 0.23 ml. TEMED 0.46 ml.
water to 100 ml. water to 100 m1.

(pH 8.9) (pH 6.7)

Stock C ; Stock D
Acrylamide 28.0 g. Acrylamide 10 g.
BIS 0.735 g. BIS 2.5 9
water to 100 ml. water to 100 m1.

Stock E Stock F
Riboflavin 4 mg. Sucrose 40 g.
water to 100 ml. water to 100 ml.

The gels were prepared by placing glass tubes

upright into rubber holders. The lower gel solution was

 

made by combining 1 part A, 2 parts C, 1 part water, and

4 parts of 0.14 g. ammonium persulfate made up to 100 ml.
with water and put into the glass tube up to 6 cm. high
using a syringe and needle. A thin layer of water was put
on the top surface to flatten it, then was removed when
polymerization was complete. The upper, small pore gel
was made of 1 part B, 2 parts D, 1 part E, and 3 parts F.
It was put on top of the polymerized lower gel to a height
of 1 cm. and the surface flattened with water. A 750 pg
portion of protein extract combined with 0.1 ml. of 40%
sucrose solution was placed on top of the gel column and
the remaining space in the glass tube filled with TRIS—
gylcine buffer, pH 8.3, diluted 1:2 with water. In the
electrophoresis apparatus, the gel tubes connected an
upper and a lower buffer reservoir, each containing TRIS-
glycine buffer. Some bromphenol blue crystals were added
to the buffer in the upper tank to enable the progress of

electrophoresis to be followed. The apparatus was taken

10 '

to a cold room at 4C where a direct current power supply,
Heathkit Model IP-32, was connected to an electrode
positioned in the center of each buffer tank, the anode
connected to the lower electrode and the cathode connected
to the upper electrode. A current of 3.5 mA per tube was
applied for the time required by the indicator dye to travel
40 mm. in the separation gel, usually one hour.

Upon completion of electrOphoresis the gel tubes

were put into crushed ice. The gels were removed from the

1‘" o-3n'.s~'~'z..-i..b .
.4.‘__- _ _

glass tubes by injecting water between the gel and the tube
wall using a syringe with a 22 gauge, 5 inch needle. They
were then put into 12% trichloracetic acid to fix the
protein. After 30 minutes the gels were stained for three
hours using comassie blue stain solution diluted 1/10 with
destain solution (Weber §E_§l., 1969). The gels were
rinsed in water and immersed in destaining solution for
two days, then stored in 12% TCA. Readings of the protein
bands were made using a Gilford 2400 spectrophotometer at
549 nm. with a gel transport attachment. Diagramatic
interpretations were made of the bands in addition to
photographs.

Location of Acid and Alkaline Phosphatase
Activity in the Gels

 

Gels taken from their glass tubes after electro-
phoresis were assayed for acid and alkaline phosphatase

activity following the methods of Jensen (1962). The gels

11

for the acid phosphatase assay were immersed directly
into a substrate solution composed of: 0.6 9. lead nitrate
in 500 ml. of 0.05 M acetate buffer pH 4.5 to which was

added sodium-beta-glycerOphosphate-5 H O in 0.10 M con-

2
centration, and the final pH adjusted to 5.0. The gels

for the alkaline phosphatase assay were immersed in a sub-
strate solution of: 5 m1. of a 2% calcium chloride solution,
2 ml. of a 2% magnesium chloride solution, 10 m1. of a 0.1 M
solution of barbital buffer and 33 ml. of water, 0.1 M

sodium-beta-glycerophosphate.5 H O; 1 N sodium hydroxide

2
was added to adjust the pH to 9.4. After incubating in
their respective solutions at 37 C for 10 minutes the gels
were rinsed in several changes of water, and in the case
of the acid phosphatase gels, once in 2% acetic acid, and
then several more times in water. Dilute sodium sulfide
solution at room temperature was used to develop the bands
to indicate where enzyme activity was located. The same
solution was also used to store the gels.
Survey for Enzymatic Activity of the
Organisms in Liquid Media and
in Protein Extracts

The liquid culture medium derived as filtrate from
shake culture flasks incubated at 24 C for seven days and
the soluble protein extracts in 0.004 M sodium bicarbonate
were tested for alkaline and acid phosphatase activity.

The enzyme substrate was paranitrophenylphosphate at a

concentration of 1 mg. per ml. in each of 0.1 M acetate

12

buffer pH 5.0 and 0.1 M TRIS buffer pH 8.5. Substrate
solution (0.8 ml.) was added to each tube plus 0.2 m1. of
culture filtrate or protein extract diluted 1:9. The
solutions were mixed well and incubated at 30 C for one
hour. Enzymatic activity was stopped using 3 m1. of

0.5 M TRIS buffer. Control tubes used for each sample
contained 0.8 m1. of water in place of substrate solution
with 0.2 ml. of the protein solution. They were treated
in the same manner as the other tubes. A Turner model 330
spectrophotometer at a setting of 410 nm was used to read
the amount of yellow colored p-nitrophenol derived in
proportion to the activity of the enzyme. The activity is

expressed as spectrophotometer O.D. (nanometers)

410
Units/one hour at 30 C.

Extracting and Analyzing
Mycelial Polysaccharides

 

 

The Gorin and Spencer (1968) method of extracting
fungal polysaccharides and analyzing the component sugars
was used for this investigation. The method of Trevelyan
gE_3;. (1950) was used to develop the chromatograms.
Polysaccharides were extracted from the mycelium using hot
potassium hydroxide. Mannan containing polysaccharides
were precipitated out of solution using Fehling solution
and then were hydrolyzed into the component sugars by acid
hydrolysis. Paper chromatography was used to identify the

sugars by running them along with known sugars on the same

.t‘é-.-n—_ so

13

Whatman 1 paper and developing them by the method noted

previously.

Requirement of Thiamine for Growth of
C. stenoceras 1013 and 1020

 

 

A total of eighteen 250 m1. Erlenmeyer flasks were
used, nine contained 50 ml. each of standard medium with
0.05 mg. of thiamine and nine flasks contained 50 ml. each
of standard medium without thiamine were used to study the

effect of thiamine on growth of the organisms. Sporothrix

 

schenckii Belo and S. stenoceras 1013 and 1020 growing on

 

 

standard agar medium were used in the experiment. Inoculum
was prepared by washing the plate cultures with sterile
buffer solution to bring the fungus spores into suspension.
Spore counts were made with a haemacytometer and the con-
centration adjusted to approximately 50x106/ml. by diluting
with sterile buffer solution. For each of the three fungi,
1 m1. of the spore suspension was put into each of six
flasks: three containing the standard medium and three
containing the medium deficient in thiamine. The flasks
were incubated at 24 C for seven days on a rotary shaker at
160 rpm. The mycelium was harvested by filtration on Seitz
sterilizing filter pads which were previously dried to
constant weight in an over at 96 C for sixteen hours and
tared. The pads plus the mycelium were dried to constant

weight as above and the weight of the mycelium calculated.

 

RESULTS

Disc Electrophoresis <1E Soluble Protein
Extracted from the Mycelium of S . schenckii ,
C. stenoceras, C. fagacearum, and
Calcarisporium arbuscula

 

 

 

 

Comparisons of the electrophoretic patterns of
soluble protein from the mycelium of seven day cultures
at 24 C were made using the eight strains: four of

S. schenckii, two of S. stenoceras, one of S. fagacearum,

 

 

 

and one of S. arbuscula. The electrophoresis patterns of
protein extract of eight strains of organisms showed
characteristic bands varying in thickness, density, and
spacing, enabling identification of the fungus source.
All eight protein extracts were tested at the same time
in six electrOphoresis runs and every run resulted in
essentially the same characteristic protein pattern.

Rf values were measured and calculated from diagramatic
drawings made directly from the gels. This method gave
Rf values as accurate as those derived from densitometer
readings and resulted in recording almost twice as many
protein bands since many were close to each other or of
low density and could not be detected from densitometer
tracings. The Rf values of corresponding bands from gels
of all six replications which were essentially the same
were totaled and an average Rf value for each band was

14

 

 

 

IllllllIl'IIIIIlllr

15

calculated, Table 1. Figure 1 shows the results of
electrOphoresis and diagramatic representations of the
average gels for the eight organisms. Average Rf values
of each protein in the electrophoretic patterns were com-
pared in all possible paired combinations to determine

the number of bands having equal Rf values for the dif-
ferent organisms and strains. The bands which were within
one Rf value of each other were considered matching. The

results of these band matchings for the organisms are

 

shown in Table 2. (j

The number of matching bands for S. stenoceras

 

1020 and three strains of S. schenckii, Pulmo, Flint, and

 

Sparrow are high, indicating a close relationship. There

are a few less for S. schenckii Belo strain. There are

 

nine matching bands between the two strains of S. stenoceras
(strains 1013 and 1020) which is less than the number of
matching bands with the other seven strains with two ‘
exceptions. The number of bands match more closely in

the imperfect stage of S. stenoceras strain 1020, to the

 

Pulmo, Flint, and Spar strains of S. schenckii than the

 

perfect stage of S. stenoceras strain 1013.

 

16

TABLE 1.--Average Rf values of protein bands (six
replicates) in the electrophoretic patterns of protein
extracts of eight organisms in standard medium for seven
days at 24 C.

 

:g.
0 ulmo

20

3O

ho

7O

50

90

100

%_' vflIW'VN‘I‘
Exam
.

I4\C-Q\1
0
£*O\FJCD trvwcrcr GDbJFJ

J
I

TITT‘YIYTITfrT‘TTTI VIII 1:

S. schenckii

g.

stenoceras

 

C.
a acearun

 

L.

AJF‘F’
O 0

CC ON
0 o

I
MN

mu bob.)
~JV1\»:D
O O O 0
box)

_1
pr
n)
O
[—4

I

11506

fi

‘51

.- It‘l—VIIIVTTurTT
U1
*0 N
o o
N ‘0

‘ l .

F7 '2
(.1-3

7L.8

I83.5

Vr—l"

 

I I

58.6

71.7

7240:;

 

 

gflintfifielo

h.h

12.5
14.?

19.1

30-7

7h.3

80.0
63.8

 

52.1

- 56.9

61.9

75.0

83.h

 

1013 “1020
2.5

u,2
7.2

9.6
11.5

16.0
22.6

25.3
28.9

31.3
BhoO
36.h
h1.7

07.3

56.2

63.0

 

 

" 2.2
6.0

9.5
12.8
16.2

20.9

20.0
27.0
29.6

3%:
32:?

39.1
h2.8

h7.h

52.9

56.0
59.4
62.3

68.9

73.h
77.8

 

C.
arbuscula

10.2
12.7

52%

 

29.3

71.1
7h.h

81.2

 

 

17

 

I .num Any .mabomsnum .w cam
Ammm Amv .Esummommmm .O “omoa Amy .maoa Amy .mcwmnum mmumoocmum .plu3onummm on

.mucoNHHom onm ADV .ucflam Any .zmmso&asm Amy “msflmuum Hflxucmnom .m mo muomuuxm
samuoum wHQSHOm Eoum mamm mUHEmHmuom ca msumuumm oaumuoamouuomamll.a musmwm

 

 

 

bum .m mom .m ONOH .m maoa .m

 

1. 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

ummm .6 onm .o ucfiam .n osasm .m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

 

 

18

TABLE 2.--The number of matching protien bands between
pairs of gels of four strains of S. schenckii, two strains
of S. stenoceras, and one each S. fagacearum, and S. arbuscula.

 

 

 

 

arb fag 1020 1013 Spar Belo Flint Pulmo

 

Pulmo 13 12 15 12 14 9 ' 15 --
Flint 11 14 14 11 15 10

Belo 10 9 11 7 9

Spar 9 13 14 11

1013 8 9 9

1020 l4 l3
fag 10

arb --

 

Tests for the Presence of Acid
and Alkaline Phospgatase
Activity in the Gels

 

 

Acid phosphatase activity for the eight strains
occurred in six of the eight gels after electrophoresis.
Two runs were made to verify the results. The gels con—

taining the proteins of S. schenckii and S. stenoceras

 

formed one to two sites of enzyme activity. All of these

gels shared a homologous site of activity while S. schenckii

 

Belo and S. stenoceras 1013 consistently exhibited a second

 

non-homologous band. The gels of S. fagacearum and S.

arbuscula had no enzyme activity. There was no evidence of

 

alkaline phosphatase activity. The results are presented

in Figure 2. To verify that the bands were the result of

19

 

l .waHm> mm AWAB auw>fluom mmmumnmmonm Uflbm
mo mmufim .AQHMV wasomsnum .0 .Amva Esnmmommmm .O .Aomoa .maoav mmuwoocmum .O
.Aummm .oamm .uswam .oEHsmv flwxoswcom .m mo mama owumnoamonuomamnl.m muamwm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

98 mg 83 23 3% 3.0 a and.“ 26:
All , I J
has .1, final.-
r 3 .31 «in 3.! .6 rl 2 I
I . I I I

 

 

20

a reaction between the protein and the enzyme substrate
solution, the gels from an electrophoretic run were incu-
bated in distilled water rather than the usual substrate
solution. No bands were evident after immersing the gels
in the sodium sulfide stain solution.

Survey for Acid and Alkaline Phosphatase

Enzyme Activities in Culture Filtrates
and Protein Extracts

 

 

 

Filtrates, collected after filtering the fungus
mycelium grown in liquid standard medium for seven days at
24 C., were tested for the presence of enzyme activity.

In Table 3 S. arbuscula filtrate showed the highest acid

 

phosphatase activity while the S. stenoceras 1020 filtrate

 

showed the lowest. The remaining filtrates were at
approximately equal levels of activity. Alkaline phos-

phatase activity was highest for S. fagacearum while

 

S. arbuscula showed a medium amount and the remaining

 

filtrates a low amount of activity. Extracts of soluble
protein from the fungus mycelium were also tested for
enzyme activity. The level of acid phosphatase activity

in the S. schenckii Belo extract was the highest while the

 

S. fagacearum extract showed the lowest. Alkaline

 

phosphatase activity was the highest for S. fagacearum

 

while S. stenoceras 1013 showed the lowest (see Table 4).

 

In general, acid phosphatase activity was higher for most
strains in both the culture filtrates and in the protein

extracts. The lower acid and alkaline phosphatase activity

TABLE 3.--Acid and alkaline phosphatase activities in the

filtrate of eight fungi grown in standard liquid medium

for seven days at 24 C.

 

Species or

O.D.

(410 nm)/ml./hour x 100

 

 

 

 

 

 

 

 

Strain Acid Phosphatase Alkaline Phosphatase
I* II III 1* II III
S. schenckii
Pulmo 28 16 12 2 4 0
Flint 44 22 0 2 4 0
Belo 32 15 18 0 3 0
Spar l8 8 17 0 2 0
S. stenoceras
1013 32 6 12 0 3 0
1020 6 4 2 0 3 0
S. fagacearum 22 15 17 -- 4 1
S. arbuscula 112 88 81 9 6 1
*
Replicate.

TABLE 4.--Acid and alkaline phosphatase activities in the
soluble protein extract of the mycelium of the eight fungi

in standard liquid medium for seven days at 24 C.

 

Species or

O.D.

(410 nm)/ml./hour x 100

 

 

 

 

 

 

 

Strain Acid Phosphatase Alkaline Phosphatase
I* II III I* II III
S. schenckii
Pulmo 8400 1435 1575 340 130 20
Flint 3480 320 4125 85 135 75
Belo 11400 4380 3930 75 80 20
Spar 1290 910 1250 50 65 20
S. stenoceras
1013 3400 2100 1700 35 40 15
1020 5775 3125 3200 380 130 120
S. fagacearum 860 635 840 2100 775 450
S. arbuscula 3250 1210 1845 820 275 180

 

 

*
Replicates.

 

22

of S. stenoceras strains 1013 and 1020 are at a level closer

to the pathogenic strains of S. schenckii than the high

 

level of enzyme activity in S. fagacearum and S. arbuscula.

 

 

Sugars Derived from Fungal
Polysaccharides

 

 

Sugar derivatives from the mycelium of S. schenckii

 

Pulmo (P) and Belo (B), and S. stenoceras 1013 (13) and

 

1020 (20) were mannose (man) and rhamnose (rh) with possible
traces of galactose (gal) and glucose (glu) while S.

fagacearum (fag) yielded mannose and glucose. These

 

results are illustrated in Figures 3 and 4. Results were
checked by co-chromatography, Figure 5, running the known
and unknown sugars together by mixing each sample with two
of the known sugars. Aside from the normal separation
pattern, any separation in the known sugar spots would
indicate that the sugars were not the same.

Effect of Thiamine Deficiency
on Growth

 

 

The results of growing S. schenckii Belo and

 

S. stenoceras 1013 and 1020 in standard medium with and

 

without thiamine at 24 C. are presented in Table 5.

Sporothrix schenckii was run as a control since it

 

requires thiamine for growth (Drouhet et a1., 1952).
Thiamine appeared to be required for the growth of

S. stenoceras strains 1013 and 1020 similar to the thiamine

 

requirement of S. schenckii.

 

23

 

gal P man 3 rh l3 9'"

3 .9.

 

Gilt»

 

Figure 3.--Paper chromatography of sugars derived
from mycelium of S. schenckii strains Pulmo (P) and Belo
(B), and S. stenoceras*1013 (13).

 

 

 

‘1'
III

24

gal 20 man My rh glu

 

Figure 4.--Paper chromatography of sugars derived
from mycelium of S. stenoceras 1020 (20) and S. fagacearum
(fag).

 

 

25

III 8: rl

 

 

Figure 5.--Paper chromatography of sugars derived
from mycelium of S. schenckii strains Pulmo (P) and Belo
(B), S. stenoceras 1013 (13) and 1020 (20), and C.

fagacearum (fag) and each sample combined with mannose
and rhamnose.

 

um—wauqlunr
, ..

26

TABLE 5.--Requirement of thiamine for growth. Weight (g.)
of mycelium from seven day liquid shake culture dried at 96 C.

 

 

 

 

 

 

S. schenckii S. stenoceras

Medium
Belo 1013 1020
Thiamine Deficient 0.002 0.000 0.031

Thiamine Enriched 0.406 0.347 0.434

 

DISCUSSION

Electrophoresis

 

Since the imperfect stage Ceratocystis stenoceras
1020 produces sporotrichosis in laboratory mice, this
strain was selected for comparison with four strains of
Sporothrix schenckii which also causes sporotrichosis.
Several isolates were used to study the extent of strain
variations. The perfect stage of S. stenoceras strain
1013 and the imperfect stage 1020 that produces sporo-
trichosis in mice, were used to compare the relationship

to S. schenckii by: disc electrophoresis, acid and

 

alkaline phosphatase activity, thiamine requirement, and
analysis of polysaccharides for their mannose contant.
Ceratocystis fagacearum was chosen for an intrageneric
comparison with S. stenoceras. Carmichael (1962) noted

that S. schenckii bears its closest resemblance to

 

Calcarisporium Preuss while S. pallidum, mentioned by

 

Barron (1968), may be more closely related to S. schenckii

than S. arbuscula and is like the "Sporotrichum" state of

 

some Ceratocystis species. Since Calcarisporium pallidum

 

was unavailable S. arbuscula was used to study the

relationships by the above mentioned methods.

27

 

28

Certain points should be mentioned as a basis for
interpreting the results of electrOphoresis of fungal
proteins. The principle of separation of proteins on
polyacrylamide gels is that the size and charge of the
protein molecules determined how far down the gel they
travel under an electrical potential. In the gels a
sieve effect is exerted on proteins due to a network of
connected strands leaving holes of a uniform size, varying
with the concentration of the acrylamide. Larger proteins
then, are retained in the upper portion of the gel while '
smaller ones are able to penetrate further. The net
electrical charge of the protein molecules in a solution
varies with the pH. The greater the charge on one of two
particles, the faster the movement in an electrical field.

Any attempt at reproduction must take into account strict
control of the pH. Considering separation of fungal
proteins with these two factors in mind, homologous proteins
may or may not occupy the same location between gels or a
band may be composed of several different proteins. A

small protein may be the same size as another, but due to
its net charge and a difference in amino acid composition,
it will not migrate as fast. It could end up forming a
homologous band or else share one with a larger protein of
greater net charge compared to other large proteins. There

are many possible variations (Ornstein, 1964).

29

Other factors affecting the results of electro-
phoresis are culture conditions of the test organism. The
age upon harvesting the fungus culture has an effect on
the pattern of protein bands (Whitney gE_Si., 1968). The
temperature, size of inoculum, and the medium must all be
the same if consistent results are to be achieved (Glynn
and Reid, 1969). In the case of this experiment all
factors appeared to be the same except for possible varia-
tion in pH and ionic strength of the TRIS-glycine buffer
used in the electrOphoresis apparatus during each run.
This was thought to have caused the slight differences
between homologOUS protein bands from one electrophoresis
run to the next. The results of the experiment could be
improved by using as many strains as possible of each

fungus, since S. schenckii showed significant differences

 

between strains. The fungi should also be compared at
several stages over the culture period and the results
averaged because Whitney EE_2$- (1968) found much variation
in percent similarity between fungi over the growing period.
The use of electrophoresis to study phylogenetic
relationships is based on the theory that the proteins
reflect the genotype. Differences in genotypes then are
supposedly shown by differences in protein and these can be
detected by electrophoresis. The degree of similarity and
difference between protein patterns of electrophoretic
gels theoretically indicates the closeness of relationship

(Dessauer and Fox, 1964).

30

The probability that proteins in homologous bands
are the same, according to Milton g£_3l. (1971), varies
directly with the closeness of relationship between the two
fungi being compared. Glynn and Reid (1969) mentioned
that it may not be valid to compare species of different

genera using electrOphoresis since a close similarity was

 

found in the protein patterns between Verticillium albo- !

atrum Reinke and Berth, and Fusarium oxysporum f. sp.

 

cubense. A larger difference was also found by Whitney
et a1. (1968) between patterns of Verticillium dahliae Ii

Kleb. and Z. albo-atrum than between 2. dahliae and

 

S. oxysporum.

 

Schechter g£_§£. (1966, 1968) obtained character-
istic protein bands from species of fungi offering
encouragement that electrophoretic analysis may be used in
the identification of fungi. However, Glynn and Reid
(1969) concluded that the method is not useful as a
taxonomic tool. In their opinion, use of electrOphoresis
in fungal taxonomic problems was made without enough con-
sideration given towards variations in the protein patterns
within a species, the limitations of the technique, and the
possible range of interpretation of the results.

Sorenson gE_§;. (1971) found variations of protein
patterns between strains of a species and concluded that
electrOphoresis may not be as valuable a taxonomic tool

as was previously thought. They derived an estimate of

31

the degree of similarity between species using coefficients
of similarity (CS) calbulated from the following formula:
Cs=a/a+b+c, where a = the number of matching bands between
the gels being compared, b = the number of bands in gel 1
which are not matching, and c = the number of bands in

gel 2 which are not matching. This method takes into
account the number of bands being compared. One of the
gels may have more bands than the other gel thereby
increasing the chance that there will be more matching
bands than a comparison between another strain with fewer
bands. This method was used to compare the results. The

figures are shown in Table 6.

TABLE 6.--Coefficients of similarity.

 

55.6 Pulmo-Flint 39.3 Belo-1020
55.6 Flint-Spar 38.2 1020-fag
53.9 Pulmo-Spar 36.5 Pulmo-fag
53.6 Pulmo-1020 35.7 Flint-Belo
50.0 Pulmo-1013 34.6 Pulmo—Belo
48.4 Spar-1020 33.3 Spar-Belo
45.2 Flint-1020 30.0 1013—1020
44.0 Spar-1013 29.1 Belo—fag
43.8 Flint-fag 29.0 1013-fag
41.9 Spar-fag 28.0 Belo-1013

40.7 Flint-1013

 

In reference to Table 6, S. schenckii strains Pulmo, Flint,
and Sparrow were approximately equal to each other in per-
cent similarity while Belo was not. This may be due to

geographic variation since Belo was isolated in Belo

32

Horizonte, Brazil while all the others were isolated in

Michigan. S. stenoceras 1013 and 1020 appeared more

 

similar to the strains of S. schenckii than to each other

 

with the exception of S. schenckii Belo. Strain 1020 was

 

alWays more similar than 1013, however. These relation-
ships are supported by the general appearance of the fungi

in culture. Strain 1020 appears more like S. schenckii

 

than 1013, forming slow growing colonies with dark pigmenta-
tion compared to the faster growing, pure white colonies
of 1013. Strain 1020, as indicated, is similar in

pathogenicity to strains of S. schenckii. S. fagacearum

 

 

followed next in its similarity to S. schenckii Flint and

 

Sparrow. The closest organism S. schenckii Belo came to

 

in similarity was S. stenoceras 1020. It was then next

 

most similar to the other three strains of S. schenckii

 

followed by S. fagacearum and S. stenoceras 1013. In View

 

 

of the findings of Glynn and Reid (1969) and Whitney

et al. (1968) the use of the results from S. arbuscula

 

in the comparisons of protein bands was deemed not valid

and was not undertaken.

Other Enzyme Studies, Sugar Analyses,
and Thiamin Requirement Studies

 

 

Using acid phosphatase activity in the gels as a
criterion, the fungi were divided into two groups: those
showing activity and those not showing activity. The gels

which showed activity were those of S. schenckii strains

 

Pulmo, Flint, Belo, and Sparrow, and C. stenoceras strains

 

33

1013 and 1020. All of the gels shared a common band while

S. schenckii Belo and S. stenoceras 1013 consistently

 

 

exhibited a second non-homologous band. All or part of the
protein in each common band must be the same since it
exhibits the same function. The lack of alkaline phosphatase
activity in the gels may have been due to dissociation of
the enzyme during electrophoresis or binding of the enzyme
to high molecular weight protein, preventing entry into
the gel.

The results of sugar analysis by paper chromatography
support those of Spencer and Gorin (1971) except for the

presence of mannose derived from S. fagacearum. They were

 

unable to obtain a mannose-containing polysaccharide from
this fungus. The reason for this difference is unknown
unless it is a strain variation. A rhamnomannan was

isolated from S. schenckii cells and used as a skin-active

 

antigen by Aoki et a1. (1968). Other polysaccharides in
fungi reported by the investigators include: mannan in

Candida, xylomannan in Cryptococcus, and galactomannan in

 

Aspergillus. The polysaccharides of each genus are dif-

 

ferent and may be valuable as a taxonomic aid.

The use of certain enzyme activities as a basis for
comparing fungi may be of some value in characterizing each
one. Large variations in results between each assay
indicates the necessity of several assays to arrive at a

more representative figure. Generally, S. schenckii and

 

34

S. stenoceras showed similar levels of activity in the

 

protein extracts and culture filtrates, while S. fagacearum

 

and S. arbuscula differed in activity.

 

Thiamine appears to be required for growth by

S. stenoceras 1013 and 1020 as well as for S. schenckii.

 

 

C. fagacearum and S. arbuscula were not tested for this

 

 

requirement. A slight increase in growth of the organisms
in the medium lacking thiamine was most likely the result
of thiamine being present in the fungal spores.

The use of various techniques, including acrylamide
gel electrophoresis to analyze the soluble protein extracted
from the eight fungi, the assay for and location of acid
and alkaline phosphatase activities, and paper chromatography
of certain sugars have been useful in showing relationships
among the organisms selected which may have taxonomic value.
Additional studies, utilizing more strains and species
should indicate even more clearly the relationships of this

complex group of organisms.

IIllIlllIlll‘ll'luI III“ I [.OI‘IIIIIIJ

SUMMARY

1. Four strains of Sporothrix schenckii; the

 

isolates of the perfect and imperfect stage of Ceratocystis

 

stenoceras; Ceratocystis fagacearum; and Calcarisporium

 

 

 

arbuscula were compared by: disc electrophoresis, enzyme

 

activity, paper chromatography, and nutritional requirement

for thiamine in the case of S. stenoceras.

 

 

2. The four strains of S. schenckii showed varia-
tions in protein bands in the electrophoretic gels. The
closeness of relationship between the fungi was evaluated
using a percentage of similarity calculated from the
number of homologous protein bands and the number of non-
homologous protein bands between paired gels. Going from
a high to a low degree of similarity: three of the four

strains of S. schenckii were similar to each other, the

 

two strains of S. stenoceras similar to S. schenckii

 

Pulmo; S. fagacearum, similar to S. schenckii Flint;

 

 

S. schenckii Belo, similar to S. stenoceras 1020; and

 

 

C. stenoceras 1013, similar to 1020. S. arbuscula was not

 

 

usable as a valid comparison since it was not, or hypothe-
sized not to be, of the same genus as the others.

3. Acid phosphatase activity was present in the
same location of the gels containing the protein of the

35

I. lllullfrlrll. . - I
Ill 4‘ II! 11‘ 4|. 4'! III. (II. 1’ .I 5!...

36

four strains of S. schenckii and the two strains of S.

 

stenoceras. No activity was detected for S. fagacearum or

 

 

S. arbuscula. There was no alkaline phosphatase activity

 

in the gels.
4. Acid and alkaline phosphatase enzyme assays in
culture filtrates at 24 C. and soluble protein extracts of

the four strains of S. schenckii and the two of S. stenoceras

 

 

showed comparable results. For most strains the acid
phosphatase activity was much higher than the alkaline

phosphatase activity. Ceratocystis fagacearum and S.

 

arbuscula showed dissimilar levels of enzyme activity.

 

5. Paper chromatography of the sugars hydrolyzed
from mannose containing polysaccharides showed the two

strains of S. schenckii and the two strains of S. stenoceras

 

 

had mannose and rhamnose sugars. Ceratocystis fagacearum

yielded mannose and glucose, while S. arbuscula had no

 

recoverable mannose.

6. The two strains of S. stenoceras produced no

 

growth or very little growth in medium deficient in thiamine
while heavy growth was evident in medium with thiamine.

The same results were obtained with S. schenckii Belo which

 

requires thiamine.
7. The results of this study support the hypothesis

of Mariat (1970) that S. schenckii is ancestrally related

 

to members of the genus Ceratocystis.

 

BIBLIOGRAPHY

37

Aoki, Y.

Barron,
D

Beneke,

Beneke,

Bievre,

BIBLIOGRAPHY

, Nakayoshi and M. Ono. 1968. Immunologically
active substances in fungi. III. The chemical
properties of skin-test antigens from Sporotrichum
schenckii, Cryptococcus neoformans, and
Aspergillus fumigatus and of serologically active
compounds in several mycotic antigens. (In
Japanese, English summary.) Arerugi. l7(l):56-6l.

 

 

G. L. 1968. The genera of Hyphomycetes from soil.
The Williams and Wilkins Co., Baltimore.

E. S., and A. L. Rogers. 19701 Medical mycology
manual. 3rd Ed. Burgess Publ. Co., Minneapolis.

E. S., R. W. Wilson, and A. L. Rogers. 1969.
Extracellular enzymes of Blastomyces detmatitidis.
Mycopath. et Myco. appl. 39:325-328.

C. de. 1967. Sur la composition proteique des
phases levuriformes et filamenteuses de Sporotrichum
schenckii etudiee par electrophorese en gel
d‘acrylamide. C. R. Acad. Sci. Ser. D. 265:537-539.

 

Carmichael, J. W. 1962. Chrysosporium and some other

 

aleuriosporic Syphomycetes. Can. J. Bot. 40:1137-
1173.

 

Carrion, A., and M. Silva. 1955. Sporotrichosis. A.M.A.

Arch. Dermatol. 72:523-524.

Davenport, A. A. 1960. Histological and histochemical

Davis,

technics. W. B. Saunders Co., Philadelphia.

B. J. 1964. Disc electrOphoresis-II. Method and
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Acad. Sci. 121, Art. 2, pp. 404-427.

Dessauer, H. S., and W. Fox. 1964. Electrophoresis in

taxonomic studies illustrated by analyses of blood
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Ed. by C. A. Leone. The Ronald Press Co., New
York, pp. 625-647.

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39

Drouhet, E., and F. Mariat. 1952. Etude des facteurs
determinant 1e develOppement de la phase levure
de Sporotrichum schencki. Ann. Inst. Pasteur.
83(4):506-514.

 

Glynn, A. N., and J. Reid. 1969. Electrophoretic patterns
of soluble fungal proteins and their possible use
as taxonomic criteria in the genus Fusarium.

Can. J. Bot. 47:1823—1831.

Gorin, P. A. J., and J. F. T. Spencer. 1968. Galacto—
mannans of Trichosporon fermentans and other
yeasts; proton magnetic resonance and chemical
studies. Can. J. Chem. 46:2299-2304.

 

Hektoen, L., and C. F. Perkins. 1900. Refractory sub—
cutaneous abscesses caused by Sporothrix‘schenckii.
A new pathogenic fungus. J. Exp. Med. 5:77-89.

 

Hughes, S. J. 1958. Revisiones Hyphomcetum aliquot cum
apendice de nominibus rejiciendis. Can. J. Bot.
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